Determination of the oral platelet aggregation inhibitor Sibrafiban in rat, dog, and human plasma utilising HPLC-column switching combined with turbo ion spray single quadrupole mass spectrometry.

A sensitive and selective HPLC-column switching method with single quadrupole mass spectrometric detection was developed for the simultaneous determination of the oral platelet aggregation inhibitor Sibrafiban (double protected prodrug), its prodrug and the active metabolite in rat, dog, and human plasma. The three analytes together with their tri-deuterated internal standards were isolated from plasma by protein precipitation (0.5 M perchloric acid). The de-proteinated samples were injected onto a standard-bore trapping column (4.0 mm i.d., LC-ABZ) of an HPLC-column switching system. Polar plasma components were removed by flushing the trapping column with ammonium formate (pH 3.6; 5 mM). Enriched compounds (including the analytes of interest) were backflushed onto a narrow-bore analytical column (2.1 mm i.d., Inertsil ODS-2) and separated by gradient elution (formic acid/ methanol). The whole effluent (200 microl/min) from the analytical column was passed to the turbo ion spray interface without splitting. Selected ion monitoring (SIM) was used for mass spectrometric detection. The limit of quantification for all three analytes was 1 ng/ml, using a 250-microl specimen of plasma. The mean precision and inaccuracy for the three analytes in all species were < 6 and < 5%, respectively. The practicability of the new analytical method was demonstrated by the analysis of about 500 rat and dog plasma and about 14,000 human plasma samples. The new method represents a successful example for the application of LC single MS with ionspray ionisation to the analysis of small molecule drugs in biological matrices from toxicokinetic studies and large clinical trials.

Simultaneous determination of a potassium channel opener and its metabolite in rat plasma with column-switching liquid chromatography using atmospheric pressure chemical ionisation.

A specific LC-MS assay was developed for simultaneous determination of Ro 31-7837 (I) and its metabolite Ro 31-6930 (II) in rat plasma, using on-line SPE by column-switching reversed-phase HPLC combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry for detection in the selected reaction monitoring mode. The method involved precipitation of plasma proteins with ethanol and automatic injection of a 1-ml aliquot of the supernatant onto a standard bore trapping column (LC-ABZ, 20x4.6 mm) for compound retention. Using the backflush mode, the analytes were transferred onto the analytical column (Kromasil C18, 125x4.0 mm) for chromatographic separation and mass spectrometric detection. The mean precision and accuracy for I and II in the concentration range 0.25-100 ng/ml were found to be 3.7% and 101%, and 3.5% and 106%, respectively. The data were assessed from QC samples during the validation phase of the assay. The lower limit of quantification for both I and II was 0.25 ng/ml, using a 0.5-ml plasma aliquot. This LC-MS method provided the requisite specificity, sensitivity, accuracy and precision to assess the pharmacokinetics of the compounds in the rat.

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching.

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.

Determination of the catechol-O-methyltransferase inhibitor Ro 40-7592 in human plasma by high-performance liquid chromatography with coulometric detection.

A sensitive and specific high-performance liquid chromatographic method has been developed to measure the catechol-O-methyl-transferase (COMT) inhibitor 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone (Ro 40-7592) in human plasma. The compound and the internal standard were extracted from plasma at pH 2 with n-butyl chloride-ethyl acetate (95:5, v/v). The extract was chromatographed on a reversed-phase column (Hypersil ODS, 5 microns) using a mixture of phosphate buffer (0.05 M, pH 2), methanol and tetrahydrofuran (45:55:5, v/v/v) as the mobile phase. Long-retained components were removed from the system by means of a simple column-switching system. Quantification of the catechol-O-methyltransferase inhibitor was performed by means of coulometric detection (0.1 V). The limit of quantification was about 1 ng/ml, using a 1-ml specimen of plasma. The recovery from human plasma was greater than 88%. The mean inter-assay precision was 5.3% in the range 2.5-1000 ng/ml. Linearity of the standard curve was obtained in the concentration range 2.5-500 ng/ml. The catechol-O-methyltransferase inhibitor was stable in human plasma when stored for six months at -20 degrees C and for 24 h at room temperature. The practicability of the new method was demonstrated by the analysis of more than 400 plasma samples from a tolerance study performed in human volunteers.

Determination of a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching.

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.

Clinical significance and hemodynamic correlates of the third heart sound gallop in aortic regurgitation. A guide to optimal timing of cardiac catheterization.

The hemodynamic and clinical data of 42 patients with chronic significant aortic regurgitation and 31 normal subjects were examined. Of the patients with aortic regurgitation, 28 had a third heart sound (S3) gallop and 14 did not. There was no significant difference in the severity of regurgitation between the patients with or without an S3 gallop. However, all patients with an S3 gallop had an abnormality increased left ventricular residual volume and depressed contractile state. These findings were supported by the hemodynamic data of two patients who underwent cardiac catheterization before and after the development of an S3 gallop. We conclude that the S3 gallop in patients with chronic AR reflects left ventricular dysfunction, rather than more severe degrees of regurgitation per se, and may therefore be useful for selecting patients for cardiac catheterization and consideration for prosthetic aortic valve replacement.

Hypercontractile cardiac state mimicking hypertrophic subaortic stenosis.

Idiopathic hypertrophic subaortic stenosis (IHSS) is a disease manifested as a spectrum of various clinical and laboratory findings. We present the case of a patient with classical clinical and hemodynamic findings of IHSS who lacked all of the typical echocardiographic features of the disease. The case emphasizes the need for diligent use of bedside physical examination and vasoactive manipulation of systolic murmurs whenever functional left ventricular outflow tract obstruction is suspected, since it can occasionally be missed by echocardiography.